Osteoporosis is a major public health problem, and it is especially prevalent in aging populations (1, 15, 21). The majority of fractures that occur in people over the age of 65 are due to osteoporosis (15, 40). Peak bone mass is a determining factor in establishing the risk of osteoporotic fracture (Heaney et al., 2000), and studies indicate that genetic factors contribute significantly to the variance in peak bone mass. One of the genes that regulate bone mass has recently been identified via positional cloning. Loss of function mutations in low density lipoprotein receptor-related protein 5 (LRP5), a co-receptor for the canonical Wnt signaling pathway (27), were found to be associated with Osteoporosis-Pseudoglioma Syndrome (OPPG), an autosomal recessive disorder which shows a reduction of bone density in humans (9). In addition, two independent kindreds that manifest familial High Bone Mass (HBM) phenotypes were found to harbor a Gly171 to Val substitution mutation (G171V) in LRP5 (5, 22). More recently, additional HBM mutations were reported in the same structural domain of the G171V mutation (36). Moreover, mice in which the LRP5 genes were inactivated by gene targeting showed phenotypes similar to those of OPPG patients (16), and transgenic expression of LRP5G171V in mice resulted in HBM (2). Furthermore, mouse primary osteoblasts showed reduced responsiveness to Wnt in the absence of LRP5 (16), and Wnt (9) or activated beta-catenin (4) stimulated the canonical Wnt signaling activity and induced the production of the osteoblast marker alkaline phosphatase (AP) in osteoblast-like cells. Together, these pieces of evidence indicate that the canonical Wnt signaling pathway plays an important role in the regulation of bone development.
Wnt
The Wnt family of secretory glycoproteins is one of the major families of developmentally important signaling molecules and has been shown to regulate a wide range of biological and pathophysiological processes that include glucose metabolism, bone remodeling, adipogenesis, neurogenesis, stem cell biology, and tumorigenesis. The canonical Wnt signaling pathway is initiated by the binding of canonical Wnts to their receptor complexes consisting of LDL receptor-related protein (LRP) 5/6 and frizzled (Fz) proteins. Through yet to be fully characterized mechanisms, beta-catenin, which is degraded via ubiquitin-mediated proteolysis in the absence of Wnts, is stabilized, leading to an increase in the cytosolic level of β-catenin. Free beta-catenin enters the nucleus and activates gene transcription in a complex with the TCF/LEF-1 transcription factors (61-66). In addition, the Wnt pathway is negatively regulated by many naturally occurring antagonists including the Dickkopf (Dkk) family of polypeptides (67, 68). Dkk binds to LRP5/6 and presumably leads to the inactivation of the receptor proteins (34). Both human and mouse genetic evidence indicates that the Wnt coreceptor LRP5 has an important role in the regulation of bone remodeling; hypomorphic or null alleles lead to early onset of osteoporosis (14), whereas different mutant alleles are associated with high bone mass phenotypes (32, 5, 51). It has been previously shown that that the mutation indirectly reduced Dkk-mediated antagonism of canonical Wnt signaling (69), thus suggesting the Dkk-LRP5 interaction as a potential therapeutic target for increasing bone mass.
Until recently, the canonical Wnt signaling pathway was believed to start when Wnt bound to frizzled Fz proteins. The seven transmembrane domain-containing Fz proteins suppress the Glycogen synthase kinase 3 (GSK3)-dependent phosphorylation of beta-catenin through ill-defined mechanisms involving Dishevelled proteins. This suppression leads to the stabilization of beta-catenin. Beta-catenin can then interact with transcription regulators, including lymphoid enhancing factor-1 (LEF-1) and T cell factors (TCF), to activate gene transcription (7, 10, 38). Recently, genetic and biochemical studies have provided solid evidence to indicate that co-receptors are required for canonical Wnt signaling in addition to Fz proteins (27, 28). The fly ortholog of LRP5/6 (LRP5 or LRP6), Arrow, was found to be required for the signaling of Wg, the fly ortholog of Wnt-1 (37). LRP5 and LRP6 are close homologues which basically function the same way, yet exhibit, different expression patterns. In addition, LRP6 was found to bind to Wnt1 and regulate Wnt-induced developmental processes in Xenopus embryos (34). Moreover, mice lacking LRP6 exhibited developmental defects that are similar to those caused by deficiencies in various Wnt proteins (30). Furthermore, LRP5, LRP6 and Arrow were found to be involved in transducing the canonical Wnt signals by binding Axin and leading to Axin degradation and beta-catenin stabilization (25, 35). The LRP5/6-mediated signaling process does not appear to depend on Dishevelled proteins (18, 31). Recently, a chaperon protein, Mesd, was identified as required for LRP5/6 transport to the cell surface (6, 11).
The involvement of the Wnt pathway in inducing repression or expansion of bone growth was demonstrated in a number of publications that described the various effects of mutations in LRP5 upon skeletal structures that served to give rise to low bone mass (14, 88) or increased bone mass (32, 5, 51). There is even a recently described genetically modified mouse model for osteoporosis, where disruption in both chromosomal copies of LRP5 (a LRP5−/− knockout) generates a low bone mass phenotype (89). However, it should be noted that even though the above mentioned references are in regard to LRP5, it should be obvious that intervention in other points along the Wnt signaling pathway could also benefit from administration of compound that have been identified through the processes of the present invention. For recent reviews of the interconnections between the Wnt pathway and bone growth, (see cited references 82, 90, and 91).
Dkk Proteins
Xenopus Dickkopf (Dkk)-1 was initially discovered as a Wnt antagonist that plays an important role in head formation (8). Thus far, four members of Dkk have been identified in mammals (17, 26). These include Dkk1, Dkk2, Dkk3 and Dkk4. Dkk1 and Dkk2 inhibit canonical Wnt signaling by simultaneously binding to LRP5 or LRP6 and a single transmembrane protein Kremen (3, 23, 24, 32). It has been previously reported that the LRP5 HBM G171V mutation appeared to attenuate Dkk1-mediated antagonism to the canonical Wnt signaling (5). The present invention describes the mechanism for this attenuation.
The third YWTD repeat domain of LRP5 which is required for Dkk-mediated antagonism of Wnt signaling has previously been identified (69). In addition, the Dkk-binding cavity and key residues within the cavity has been delineated by site-directed mutagenesis (69). This cavity is located at the large opening of the barrel-like structure of the YWTD repeat domain that is made of six beta-propellers (FIG. 17A). Importantly, the two most important residues in the interaction with Dkk, Residues Glu721 and Trp780 (69), are located at the bottom of this cavity, suggesting that small molecule chemicals that bind to this cavity may be able to disrupt the Dkk-LRP5 interaction by blocking the access to this key residue.